STRUCTURAL VARIATION AMONG RETROVIRAL PRIMER-DNA JUNCTIONS - SOLUTIONSTRUCTURE OF THE HIV-1 (-)-STRAND OKAZAKI FRAGMENT R(GCCA)D(CTGC)CENTER-DOT-D(GCAGTGGC)

The three-dimensional solution structure of the hybrid-chimeric duplex r(gcca)d(CTGC). d(GCAGTGCC) has been determined by two-dimensional NM R, restrained molecular dynamics (rMD), and NOE back-calculation metho ds, This chimera, consisting of a chimeric RNA-DNA strand and its comp lementary DNA strand, is formed after priming (-)-strand DNA synthesis by tRNA(Lys3) and subsequent (+)-strand DNA synthesis by reverse tran scriptase and is an obligatory intermediate in the formation of double -stranded DNA prior to HIV-1 retrovirus integration. The duplex consis ts of two different types of double helix: a hybrid form (H-form) and a B-form structure connected by a junction. It is chemically similar t o several other Okazaki fragments whose structures have been previousl y determined in our laboratory. However, some structural parameters ar e not the same and were found to be sequence dependent. In particular, the sugar conformations at the DNA base pair proximal to the hybrid s egment vary from O4'-endo to C2'-endo depending on the base compositio n. The position of the transition from the relatively wide groove of H -form to the narrow groove of B-form is also sequence dependent, occur ring either exactly at the RNA-DNA junction or within the purely DNA s egment of the chimera-as is the case in the structure of the present H IV-1 (-)-strand primer. This structural change produces a kink at the DNA-DNA step adjacent to the RNA-DNA junction in the HIV-1 (-)-strand primer. The sequence dependence of structures of RNA-DNA chimeric dupl exes may be responsible for the variable cleavage pattern of different Okazaki fragments by reverse transcriptase RNase H.